2C). IB analysis revealed that transient or stable silencing of endogenous RACK1 expression

by RACK1 small interfering RNA (siRNA) or short hairpin RNAs (shRNAs) in HepG2 cells significantly suppressed basal levels of P-JNK. Reduced P-JNK levels under the condition of RACK1 knockdown were associated with decreased P-MKK7 levels (Fig. 3A-C). Similar phenomena were also observed in Huh7 and SK-Hep-1 cells (Fig. 3B). By contrast, transient ectopic expression of RACK1 in HepG2 cells led to substantially enhanced basal levels of both P-JNK and P-MKK7 (Fig. 3D). Moreover, single-clone HepG2 stable transfectants (named FLAG-RACK1Low and FLAG-RACK1high, respectively, according to levels of FLAG-RACK1 protein) also exhibited augmented levels of

P-JNK and P-MKK7, which were well selleckchem correlated with FLAG-RACK1 expression (Fig. 3E). These data collectively indicate that RACK1 contributes to enhanced levels of P-MKK7/P-JNK in human HCC cells. MKK7 is composed of an N-terminal JNK-binding domain and a kinase domain, SB203580 chemical structure whereas RACK1 contains seven Trp-Asp (WD) repeats.14, 15, 20 RACK1/MKK7-interacting regions were analyzed through generating several deletion mutants (Fig. 4A), followed by Co-IP analysis in 293T cells. FLAG-RACK1 coprecipitated with coexpressed kinase domain of MKK7 (MKK7

ΔN), but not with coexpressed JNK-binding domain of MKK7 (MKK7 ΔC) (Fig. 4B). On the other hand, GFP-MKK7 coprecipitated with coexpressed RACK1 deletion mutant that included WD domains five to seven (RACK1 WD5-7), but not with coexpressed RACK1 WD1-4 (Fig. 4C). Furthermore, a WD6- or WD7-truncated RACK1 mutant (FLAG-WDΔ6 or FLAG-WDΔ7), but not FLAG-WDΔ5, showed significant reduced association check details with coexpressed GFP-MKK7 (Fig. 4D). WD6 and WD7 of RACK1 are the docking domains for various proteins, including MEKK4.14, 15 To analyze whether the direct interaction between RACK1 and MKK7 enhances the activity of the JNK pathway, it is of importance to identify the specific binding sites in RACK1. In this scenario, molecular simulations of MKK7 and RACK1 were performed according to the reported three-dimentional crystal structures of the proteins (Supporting Fig. 2A), followed by molecular docking. Among the candidates for the complex structure, the one with WD6 and WD7 in the interfaces was chosen. The predicted model suggested that three previously unidentified sites (amino acids 225-231 in WD6 and amino acids 269-272 and 275-280 in WD7) of RACK1 were essential for anchoring the kinase domain of MKK7 (Supporting Fig. 2A).